: Bacterial DNA replication is carefully controlled at the initiation stage, possibly by regulation of the t essential activity of IIJnaA protein. The cellular membrane has long been hypothesized to be involved in chromosomal replication, with accumulating evidence that indicates membranes have a profound influence on DnaA protein. E. coli membrane liposomes can convert an inert form of purified DnaA protein into a replicatively active form. Characterization of the membranes has identified fluidity and acidic phospholipids as essential features. The importance of membrane fluidity and acidic phospholipids for this activation has been seen in vitro, and for DNA replication, in vivo. The long-term goal of this research is to elucidate the physiological significance of the influence of membranes on chromosomal replication. The research outlined here uses biochemical approaches with defined components, genetic and physiological studies, and cytolocalization techniques to directly test the hypothesis that the cellular membrane participates in the regulation of DnaA protein activity. The Specific Aims are to: Determine if the localizations of DnaA and the chromosomal origin to the site of initiation occur dependently or independently of each other, and if their proper localizations require acidic phospholipids. Using fluorescence microscopy, examine the importance of DnaA and acidic phospholipids in the proper localization of the chromosomal origin, and the importance of an intact origin and acidic phospholipids in the proper localization of DnaA protein. Generate strains in which the sole allele of dnaA is located on the chromosome and encodes for DnaA proteins with altered membrane-associating properties. Analyze how the mutant DnaA proteins affect cell cycle controlled chromosomal replication in vivo. Map the mutations in dnaA and examine the replication activities of the mutant proteins in vitro. Determine how the cellular locations of the mutant DnaA proteins may differ from that of wild-type DnaA protein. Isolate and identify the membrane component that inhibits acidic phospholipid reactivation of inert ADP-DnaA to active ATP-DnaA protein. Characterize the effect that deletion and overexpresslon of the gene(s) that encode the nucleotide release inhibitor have on the cell cycle controlled initiation of replication in vivo. This work should provide insight into the regulation of the initiation of DNA replication, which is a key control point in the prokaryotic cell cycle and in the deterniination of eukaryotic cellular quiescence or proliferation. Furthermore, knowledge gained from the proposed studies of DnaA protein may guide future investigations of how phosphohpids act as regulators of enzymatic activities.